Methods, Devices, and Apparatus for Washing Samples on Array Plates

ABSTRACT

A method for washing particles includes obtaining an array plate that includes an array of hydrophilic areas surrounded by one or more hydrophobic areas. A respective solution containing a sample is located on a respective hydrophilic area of the array of hydrophilic areas. The respective hydrophilic area includes one or more indentations from a respective surrounding hydrophobic area of the one or more hydrophobic areas. The respective hydrophilic area includes a first indented surface that is offset from a reference surface defined by the respective surrounding hydrophobic area. The method also includes placing an aspirator nozzle above the respective hydrophilic area at a predefined distance from the first indented surface, and aspirating the solution with the aspirator nozzle while the aspirator nozzle is located at the predefined distance from the first indented surface.

RELATED APPLICATIONS

This application is a continuation application of Ser. No. 16/590,187,filed Oct. 1, 2019, which is a continuation application of InternationalPatent Application No. PCT/IB2018/000436, filed Apr. 5, 2018, whichclaims the benefit of, and priority to, U.S. Provisional PatentApplication Ser. No. 62/482,140, filed Apr. 5, 2017, U.S. ProvisionalPatent Application Ser. No. 62/517,166, filed Jun. 9, 2017, and U.S.Provisional Patent Application Ser. No. 62/517,788, filed Jun. 9, 2017.All of these applications are incorporated reference herein in theirentireties.

TECHNICAL FIELD

The disclosed embodiments relate generally to methods, devices, andapparatus for washing samples (e.g., cells, particles, etc.). Moreparticularly, the disclosed embodiments relate to methods, devices, andapparatus for washing samples on array plates and slides.

BACKGROUND

An array plate is also called a microtiter plate, microplate, ormicrowell plate. Array plates are typically used to hold respectiveliquid droplets separately for biological and/or chemical reaction. Forexample, a well-type array plate includes a plurality of wells so thateach liquid droplet or each sample may be dispensed into a separate wellfor further processing. Typically, the number of wells is selected from6, 24, 96, 384, 1536, 3456, and 9600.

Samples (e.g., cells) are frequently washed. Washing typically involvesadding a wash solution to a sample solution, including samples (e.g.,cells), on the slide and removing the mixture of the wash solution andthe sample solution. By repeating the dilution and partial removal ofthe sample solution, the concentration of chemicals and/or biologicalreagents other than the samples are reduced. However, certain cells(e.g., suspension cells, non-adherent cells, and weakly adherent cells)do not strongly adhere to the slide. Thus, during removal of themixture, cells may be removed along with the mixture, thereby reducingthe number of cells that remain on the hydrophilic area of the slideafter the washing. Because a reliability of cell-based reactionstypically requires a sufficient number of cells, the loss of cellsduring washing negatively affects cell-based reactions.

In addition, variations in the sample washing increase measurementerrors, which are not desirable for accurate assays.

SUMMARY

Accordingly, there is need for methods, devices, and apparatus thatbetter retain cells during washing. Such methods, devices, and apparatusplates may replace the conventional methods, devices, and apparatus forwashing cells. Such methods, devices, and apparatus reduce or eliminatethe loss of cells during washing, thereby improving the reliability ofcell-based reactions. Similarly, such methods, devices, and apparatusmay be used in washing other types of samples, such as beads orparticles conjugated with target molecules. In addition, such methods,devices, and apparatus improve the accuracy in assays and reduce thetime required for washing samples.

A number of embodiments that overcome the limitations and disadvantagesof existing methods, devices, and apparatus are presented in more detailbelow. These embodiments provide methods, devices, and apparatus forwashing a sample in a solution.

As described in more detail below, in accordance with some embodiments,an apparatus for washing an array plate includes one or more dispensers.A respective dispenser of the one or more dispensers is configured todispense a first liquid on the array plate. The respective dispenserincludes a first piston configured to slide at least partially within afirst channel; and a first valve configured to allow the first liquid inthe first channel to be dispensed from the first channel through thefirst valve and prevent a liquid from entering into the first channelthrough the first valve.

In accordance with some embodiments, an apparatus for washing an arrayplate includes one or more aspirators. A respective aspirator of the oneor more aspirators is configured to aspirate a liquid on an array plate.The respective aspirator includes a piston configured to slide at leastpartially within a channel; and a valve configured to allow the liquidon the array plate to be aspirated into the channel through the valveand prevent a liquid in the channel from exiting from the channelthrough the valve.

In accordance with some embodiments, an apparatus for washing an arrayplate includes one or more dispensers, a respective dispenser of the oneor more dispensers configured to dispense a first liquid on the arrayplate; and one or more aspirators that are distinct from the one or moredispensers, a respective aspirator of the one or more aspiratorsincluding a positive displacement pump configured to aspirate liquid onthe array plate.

In accordance with some embodiments, a method for washing a sampleincludes obtaining an array plate that includes an array of hydrophilicareas surrounded by one or more hydrophobic areas. A respective solutioncontaining a sample is located on a respective hydrophilic area of thearray of hydrophilic areas. The respective hydrophilic area includes oneor more indentations from a respective surrounding hydrophobic area ofthe one or more hydrophobic areas. The respective hydrophilic areaincludes a first indented surface that is offset from a referencesurface defined by the respective surrounding hydrophobic area. Themethod also includes placing an aspirator nozzle above the respectivehydrophilic area at least 100 μm from the first indented surface; andaspirating the solution with the aspirator nozzle while the aspiratornozzle is located at least 100 μm from the first indented surface.

In accordance with some embodiments, an apparatus is configured forperforming any method described herein.

In accordance with some embodiments, a device for washing a sampleincludes a plate having an array of hydrophilic areas; and one or morehydrophobic areas surrounding the array of hydrophilic areas. Arespective hydrophilic area of the array of hydrophilic areas is offsetfrom a surrounding hydrophobic area of the one or more hydrophobicareas. The respective hydrophilic area includes a primary area and twoor more secondary areas that extend from the primary area on a planedefined by the primary area.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the aforementioned embodiments as well asadditional embodiments, reference should be made to the Description ofEmbodiments below, in conjunction with the following drawings in whichlike reference numerals refer to corresponding parts throughout thefigures.

FIGS. 1A-1F illustrate a washing operation with a conventionalmicro-titer plate.

FIGS. 2A-2E illustrate washing operations with an array plate having ahydrophilic region and a hydrophobic area in accordance with someembodiments.

FIGS. 3A-3G illustrate a washing operation in accordance with someembodiments.

FIG. 3H illustrates components of a washing apparatus in accordance withsome embodiments.

FIGS. 4A-4C are perspective views of array plates in accordance withsome embodiments.

FIGS. 4D-4F are partial plan views of example array plates in accordancewith some embodiments.

FIGS. 4G-4H illustrate arrangement of dispensers and aspirators inaccordance with some embodiments.

FIGS. 5A-5I are partial cross-sectional views of example array plates inaccordance with some embodiments.

FIGS. 5J-5K illustrate an example array plate in accordance with someembodiments.

FIG. 5L illustrates an array plate in accordance with some embodiments.

FIG. 5M illustrates an array plate in accordance with some embodiments.

FIG. 5N illustrates a grid configured for use with an example arrayplate in accordance with some embodiments.

FIG. 6A shows results of washing operations performed with aconventional micro-titer plate in accordance with some embodiments.

FIG. 6B shows results of washing operations performed with an examplemethod described herein.

FIG. 6C illustrates the results of fluorescence-activated cell sortingof lymphocytes washed using a conventional method in accordance withsome embodiments.

FIG. 6D illustrates the results of fluorescence-activated cell sortingof lymphocytes washed using the method described herein in accordancewith some embodiments.

FIG. 7A shows results of washing operations performed with an arrayplate without filleted corners in accordance with some embodiments.

FIG. 7B shows results of washing operations performed with an arrayplate with filleted corners in accordance with some embodiments.

FIG. 7C shows results of washing operations performed with an arrayplate without filleted corners in accordance with some embodiments.

FIG. 7D shows results of washing operations performed with an arrayplate with filleted corners in accordance with some embodiments.

Like reference numerals refer to corresponding parts throughout thedrawings.

DESCRIPTION OF EMBODIMENTS

Methods, devices, and apparatus for washing samples are described.Reference will be made to certain embodiments, examples of which areillustrated in the accompanying drawings. While the claims will bedescribed in conjunction with the embodiments, it will be understoodthat it is not intended to limit the claims to these particularembodiments alone. On the contrary, the embodiments are intended tocover alternatives, modifications and equivalents that are within thespirit and scope of the appended claims.

Moreover, in the following description, numerous specific details areset forth to provide a thorough understanding of the embodiments.However, it will be apparent to one of ordinary skill in the art thatthe embodiments may be practiced without these particular details. Inother instances, methods, procedures, components, and networks that arewell-known to those of ordinary skill in the art are not described indetail to avoid obscuring aspects of the embodiments.

It will also be understood that, although the terms first, second, etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. For example, a first piston could be termed asecond piston, and, similarly, a second piston could be termed a firstpiston, without departing from the scope of the embodiments. The firstpiston and the second piston are both pistons, but they are not the samepiston.

The terminology used in the description of the embodiments herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe embodiments and the appended claims, the singular forms “a,” “an,”and “the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. It will also be understood that theterm “and/or” as used herein refers to and encompasses any and allpossible combinations of one or more of the associated listed items. Itwill be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, integers, steps, operations, elements, and/orcomponents, but do not preclude the presence or addition of one or moreother features, integers, steps, operations, elements, components,and/or groups thereof.

As used herein, a liquid droplet refers to an aliquot of a liquid. Adroplet may have any shape, and the term “droplet” is not used herein todescribe a particular shape.

FIGS. 1A-1F illustrate a washing operation with a conventionalmicro-titer plate.

FIG. 1A illustrates solution 104 containing samples 114 (e.g., cells,particles, etc.) in a well that is defined in micro-titer plate 102.

FIG. 1B illustrates that dispenser 110 containing wash liquid 106 (e.g.,a wash buffer, such as phosphate-buffered saline, Tris-buffered saline,borate-buffered saline, and TE buffer) is used for washing samples 114.

For example, as shown in FIG. 1C, wash liquid 106 in dispenser 110 isdispensed into solution 104, thereby forming mixture 108 (e.g., liquid)of solution 104 and wash liquid 106. As a result, chemical andbiological reagents in solution 104 are diluted (e.g., concentrations ofchemicals and biological reagents in solution 104 are reduced). FIG. 1Calso illustrates that at least a portion of samples 114 is lift-off fromthe bottom of the well and suspended in mixture 108, due to the liquidflow caused by introduction of wash liquid 106 into solution 104.

FIG. 1D illustrates that samples 114 settle over time.

FIG. 1E illustrates that aspirator 120 is used to aspirate (e.g.,remove) a portion of mixture 108.

FIG. 1F illustrates that aspirator 120 has aspirated a portion ofmixture 108. The volume of mixture 108 remaining in the well defined inmicro-titer plate 102, after the portion of mixture 108 is aspirated, isdetermined at least in part by height V of aspirator 120 (e.g., adistance between a nozzle tip of aspirator 120 and a bottom of the welldefined in the micro-titer plate 102).

FIG. 1F also illustrates that a portion of samples 114 is also aspiratedby aspirator 120. Wells of micro-titer plate 102 have a high aspectratio (e.g., a ratio between the height of the well and the diameter ofthe well). Thus, once samples 114 are agitated, it takes a long time forsamples 114 to settle down. If a portion of mixture 108 is aspiratedbefore samples 114 have fully settled down, a portion of samples 114that is aspirated is increased.

In addition, FIG. 1F illustrates that samples 114 cluster toward cornersof the well when the volume of mixture 108 is reduced. In addition,mixture 108 clings toward corners of the well. Both of these can reducethe efficiency of washing.

FIGS. 2A-2E illustrate washing operations with an array plate having ahydrophilic region and a hydrophobic area in accordance with someembodiments.

FIG. 2A is a partial cross-section of an array plate, where hydrophilicregion 204 is surrounded by hydrophobic area 206. In FIG. 2A, solution104 containing samples 114 is located over hydrophilic region 204.Solution 104 is retained over hydrophilic region 204, as surroundinghydrophobic area 206 prevents spreading of solution 104 beyondhydrophilic region 204.

FIG. 2A also illustrates dispenser 210 and aspirator 220. Dispenser 210includes liquid 106 for washing samples 114 in solution 104 (by dilutionof solution 104).

The array plate illustrated in FIG. 2A is configured to hold solution104 without tall side walls, like conventional micro-titer plates. Thus,in the configuration shown in FIG. 2A, there are no corners toward whichsolution 104 and samples 114 cluster.

In addition, solution 104 in FIG. 2A has a low aspect ratio (e.g., aratio between the height of solution 104 and the width or diameter ofsolution 104 on the array plate is less than the height of solution 104and the diameter of solution 104 in a conventional micro-titer plate,sometimes by a factor of 2, 4, 6, 8, 10, or 20). Thus, when samples 114are agitated, samples 114 in solution 104 on the array plate can settlefaster than samples in solution 104 in a conventional micro-titer plate(shown in FIG. 1F).

In some embodiments, magnetic particles configured to couple with cells(e.g., coated with materials that can reversibly or irreversibly bind tothe cells) are included in solution 104 (e.g., by introducing themagnetic particles into solution 104). Once the magnetic particles bindto the cells in solution 104, a magnetic field is applied to themagnetic particles in solution 104 to accelerate settling of themagnetic particles (and associated cells).

The inventors of this application have also discovered that the distancebetween hydrophilic region 204 and aspirator 220 (e.g., a distancebetween hydrophilic surface 204 and a nozzle tip of aspirator 220) isimportant in improving retention of samples 114. In some embodiments,aspirator 220 needs to be positioned at least 100 μm from hydrophilicregion 204. In some embodiments, aspirator 220 needs to be positioned atleast 200 μm from hydrophilic region 204. In some embodiments, aspirator220 needs to be positioned at least 300 μm from hydrophilic region 204.

FIG. 2B illustrate dispenser 210 and aspirator 220 with improved volumecontrol. A variation in the dispensed volume and/or the aspirated volumecontributes to a variation in the dilution factor, which leads to anincreased error in assays. Thus, reducing the variation in the volume ofthe dispensed liquid and/or the volume of the aspirated liquid improvesthe assay accuracy (e.g., an accuracy of an assay performed using thewashing operation).

In FIG. 2B, dispenser 210 includes valve 212 (e.g., a one-way valve,which is also called a check valve, or a check valve) to reduce thevariation in the volume of the dispensed liquid, and aspirator 220includes valve 222 (e.g., a one-way valve or a check valve) to reducethe variation in the volume of the aspirated liquid. For example, arespective valve allows a liquid to flow in one direction but preventsthe liquid to flow in the opposite direction (e.g., valve 212 allows theliquid in dispenser 210 to exit from dispenser 210 through valve 212 butprevents a liquid to enter into dispenser 210 through valve 212, andvalve 222 allows mixture 108 to enter into aspirator 220 through valve222 but prevents mixture 108 in aspirator 220 from exiting fromaspirator 220 through valve 222).

FIG. 2C is similar to FIG. 2B, except that dispenser 230 is used inplace of dispenser 210 and aspirator 240 is used in place of aspirator220. Dispenser 230 includes piston 232 (e.g., a plunger) configured toslide within channel 234 for dispensing wash liquid 106 in channel 234through valve 212. Aspirator 240 includes piston 242 (e.g., a plunger)configured to slide within channel 244 for aspirating a liquid (mixture108) into channel 244 through valve 222. In some embodiments, channel234 is defined by tube 235. In some embodiments, channel 244 is definedby tube 245.

In some implementations, the volume of the aspirated liquid iscontrolled by a movement of piston 242 (e.g., a diameter of channel 244and a travel distance of piston 242). In some embodiments, the diameterof piston 242 is less than the diameter of mixture 108, whichfacilitates an accurate control of the volume of the aspirated solution.Similarly, the volume of the aspirated liquid is accurately controlledby a movement of piston 232. In some implementations, the volume of theaspirated liquid (and/or the remaining liquid) is determined based on aheight of an aspirator (e.g., a portion of the liquid located above thetip of aspirator 240 is aspirated and a portion of the liquid locatedbelow the tip of aspirator 240 remains, as shown in FIG. 1F).

FIG. 2D is similar to FIG. 2C, except that piston 236 defines channel238 within piston 236 and piston 236 is coupled with valve 252 (e.g., aone-way valve, a check valve, etc.), and piston 246 defines channel 248within piston 246 and piston 246 is coupled with valve 262 (e.g., aone-way valve, a check valve, etc.). Channel 238 and valve 252 areconfigured to deliver a precise volume of wash liquid 106 into channel234. Channel 248 and valve 262 are configured to remove mixture 108 inchannel 244. The operations of these components are described furtherbelow with respect to FIGS. 3A-3G.

FIG. 2E is similar to FIG. 2D, except that filter 250 is coupled with atip of aspirator 240. In some implementations, filter 250 reduces orprevents aspiration of cells. In some embodiments, filter 250 has aplurality of pores. In some embodiments, the plurality of pores has apore size between 0.1 and 20 μm. In some embodiments, the plurality ofpores has a pore size between 1 and 10 μm. In some embodiments, theplurality of pores has a pore size between 1 and 5 inn. In someembodiments, the plurality of pores has a pore size between 2 and 8 μm.

FIG. 2E also illustrates that aspirator 240 is coupled with vibrator254. In FIG. 2E, vibrator 254 is positioned adjacent to filter 250.Vibrator 254 is configured to provide vibration to filter 250, whichreduces clogging of filter 250 by preventing accumulation of cells onfilter 250. In some embodiments, vibrator 254 is a piezo-electricvibrator.

FIG. 3A illustrates that dispenser 230 includes piston 236 in a firstposition. The channel defined within piston 236 includes wash liquid106.

FIG. 3B illustrates that piston 236 moves up to a second position, whichallows liquid 106 in the channel defined within piston 236 to flow intochannel 234. During the upward movement of piston 236, there is anegative pressure within channel 234, which keeps valve 212 closed.

Once channel 234 is filled with a predefined volume of wash liquid 106,piston 236 moves down to push wash liquid 106 out of channel 234. FIG.3C illustrates that piston 236 moves down, which causes valve 252 toclose. The increased pressure within channel 234 opens valve 212 so thatwash liquid 106 in channel 234 is dispensed (e.g., released) into samplesolution 104, thereby forming mixture 108.

FIG. 3D illustrates that piston 236 has returned to the first position.In FIG. 3D, piston 246 of aspirator 240 is in a third position.

FIG. 3E illustrates an upward movement of piston 246 to a fourthposition. The negative pressure within channel 244 causes valve 222 toopen, which allows a portion of mixture 108 to flow into channel 244.The negative pressure within channel 244 causes valve 262 to close sothat mixture 108 does not flow into the channel 248.

Once channel 244 is filled with a predefined volume of mixture 108,piston 246 moves down to move mixture 108 in channel 244 to channel 248.FIG. 3F illustrates piston 246 moves down, which causes valve 222 toclose. The increased pressure within channel 244 opens valve 262 so thatmixture 108 in channel 244 flows into channel 248.

FIG. 3G illustrates that piston 246 has returned to the third position.

In some embodiments, dispenser 230 is coupled with a wash liquid source(e.g., a reservoir containing a wash liquid, which is optionallycombined with a pump configured to provide the wash liquid). Forexample, wash liquid 106 is provided to channel 238 by the wash liquidsource. In some embodiments, aspirator 240 is coupled with a suctionpump. For example, mixture 108 in channel 248 is removed by the suctionpump. In some embodiments, aspirator 240 is coupled with a reservoir.For example, mixture 108 in channel 248 is drained to the reservoirwhile piston 246 moves up.

In some embodiments, subsequent to dispensing wash liquid 106 and priorto aspirating a portion of mixture 108, mixture 108 is shaken and/oragitated (e.g., the array plate on which mixture 108 is located isshaken and/or agitated by placing the array plate on a shaker andactivating the shaker).

In some embodiments, one or more valves illustrated in FIGS. 3A-3G(e.g., valves 212, 222, 252, and 262) are spring-loaded. A spring-loadedvalve is configured to close itself and/or remain closed when a pressuredifference applied on the valve is less than a predefined threshold.

Although FIGS. 3A-3G illustrate that a single dispenser and a singleaspirator for a single sample spot, in some embodiments, multipledispensers and/or multiple aspirators are used for a single sample spot(e.g., using multiple dispensers and multiple aspirators for aparticular sample spot can reduce the washing time, especially for alarge sample spot). In some embodiments, multiple dispensers areconfigured for concurrent operations and/or multiple aspirators areconfigured for concurrent operations. For example, multiple dispensersare built into a single block, and multiple aspirators are built into asingle block, as shown in FIG. 3H.

In some embodiments, a single dispenser is used for dispensing a washliquid into multiple spots. For example, a single dispenser is coupledwith a split channel (e.g., 2-channel, 4-channel, 8-channel, 12-channel,16-channel, 32-channel, 64-channel, 128-channel, 256-channel splitter).In some embodiments, a single aspirator is used for aspirating liquid(e.g., a mixture) from multiple spots. For example, a single aspiratoris coupled with a split channel (e.g., 2-channel, 4-channel, 8-channel,16-channel, 32-channel, 64-channel, 128-channel, 256-channel splitter).

In some embodiments, one or more of a dispenser and an aspirator arecoupled with a positive displacement pump (e.g., a membrane pump, suchas a solenoid micropump). The positive displacement pump reduces thevariation in the volume of the dispensed liquid or the volume of theaspirated liquid. In some embodiments, a dispenser is coupled with apositive displacement pump without a valve. In some embodiments, anaspirator is coupled with a positive displacement pump without a valve.

Although FIGS. 2A-2E and 3A-3G illustrate configurations, in which botha dispenser and an aspirator are concurrently in contact with a liquid(e.g., solution 104 or mixture 108), a person having ordinary skill inthe art would understand that only one of the dispenser and theaspirator may be in contact with the liquid (e.g., a dispenser comes incontact with solution 104 first for dispensing a wash liquid, while anaspirator remains separated from solution 104, and the dispenser issubsequently removed from mixture 108 of solution 104 and the washliquid, and the aspirator comes in contact with mixture 108 foraspirating a portion of mixture 108 while the dispenser remainsseparated from mixture 108). In some embodiments, a dispenser is used ata first time without an aspirator, and an aspirator is used at a secondtime distinct from the first time (e.g., the second time is subsequentto the first time) without a dispenser. For brevity, these details areomitted.

In FIGS. 1A-1F, 2A-2E, and 3A-3G, top portions of dispensers andaspirators are truncated to simplify the drawings.

Although FIGS. 2A-2E and 3A-3G illustrate washing operations, analogousoperations can be used for introducing reagents to the array plate (orthe cells on the array plate). For example, instead of a wash liquid, areagent liquid (e.g., a liquid containing reagents for reaction withcells) is used in some implementations. Such operations can introducethe reagents without agitating the cells on the array plate, therebyimproving the accuracy and reliability of reaction between the reagentsand the cells. In addition, the loss of the cells is reduced by usingsuch operations.

Although FIGS. 2A-2E and 3A-3G illustrate an aspirator located away froma dispenser (e.g., the aspirator and the dispenser are located towardtwo opposite ends of solution 104), in some implementations, theaspirator and the dispenser are located adjacent to each other (e.g.,the aspirator and the dispenser are located toward a same end ofsolution 104, or toward the center of solution 104).

FIGS. 4A-4C are perspective views of array plates in accordance withsome embodiments.

FIG. 4A illustrates array plate 400 with base 420. On top of base 420,hydrophilic regions 412 (e.g., 412-1, 412-2, 412-3, 412-4, 412-5, 412-6,412-7, and 412-8) are surrounded by hydrophobic area 410.

FIG. 4B illustrates array plate 402, which is similar to array plate 400except that hydrophilic regions 412 are offset from surroundinghydrophobic area 410.

In some embodiments, a respective hydrophilic region 412 (e.g.,hydrophilic region 412 in FIG. 4A or FIG. 4B) has a circular shape or anellipsoidal shape.

FIG. 4C illustrates array plate 404, which is similar to array plate 400except that hydrophilic regions 412 (called herein “primary areas”) arecoupled with one or more secondary areas, such as secondary areas 414(e.g., 414-1) and 416 (e.g., 416-1). Although each hydrophilic region412 in FIG. 4C is coupled with two secondary areas, a respectivehydrophilic region may have only one secondary area, or more than twosecondary areas (e.g., three or four secondary areas). A secondary areais a hydrophilic region that is configured for placing a dispenserand/or an aspirator. However, in some embodiments, a hydrophilic regionwithout any secondary area (e.g., array plates 400 and 402 shown inFIGS. 4A and 4B) is used, and a dispenser and an aspirator arepositioned over the hydrophilic region (e.g., a primary area).

FIGS. 4D-4F are partial plan views of example array plates in accordancewith some embodiments. In FIGS. 4D-4F, a respective hydrophilic region(or a primary area) has a circular shape, and a respective secondaryarea has a shape that corresponds to a portion of a circle (e.g., acrescent shape).

FIG. 4D illustrates that a hydrophilic region (also called herein aprimary area) and secondary areas are located on a same plane.

FIG. 4E illustrates that a hydrophilic region (or a primary area) islocated on a plane different from a plane on which secondary areas arelocated. Partial cross-sections of different embodiments that correspondto FIG. 4E are illustrated in FIGS. 5A-5D.

FIG. 4F illustrates a hydrophilic region (or a primary area) with fouradjacent secondary areas in accordance with some embodiments.

FIGS. 4G-4H illustrate arrangement of dispensers and aspirators inaccordance with some embodiments.

FIG. 4G illustrates that a row of dispensers 210 and a row of aspirators220 are used. As shown in FIG. 4G, a row of dispensers 210 is used todispense a wash liquid to a row of spots (or associated hydrophilicsecondary areas) and a row of aspirators 220 is used to aspiratemixtures from the same row of spots (or associated hydrophilic secondaryareas). In some cases, after a row of spots is washed, the array plateand/or the dispensers and the aspirators are moved so that the next rowof spots can be washed.

FIG. 4H illustrates that a two-dimensional array of dispensers 210 and atwo-dimensional array of aspirators 220 are used.

FIG. 5A illustrates a partial cross-section of array plate 520. Arrayplate 520 includes base 502 with secondary areas 514 and 516 located ona same plane as surrounding hydrophobic area 506. Primary area 512 islocated offset from the plane on which secondary areas 514 and 516 arelocated (e.g., primary area 512 is indented so that primary area 512 islocated deeper than secondary areas 514 and 516).

In some embodiments, the hydrophilic areas (e.g., the primary areaand/or the secondary areas) include, or are made of, hydrophilicmaterials, such as polyvinyl alcohol, poly vinyl pyrrolidone, etc. Insome embodiments, the hydrophobic area includes, or is made of,hydrophobic materials, such as a polytetrafluoroethylene (PTFE) matrix,poly(methyl-methacrylate), etc.

In some embodiments, the hydrophilic areas (e.g., the primary areaand/or the secondary areas) include, or are made of, glass (e.g., theprimary area and/or the secondary areas are etched into glass). In someembodiments, the hydrophobic area includes a layer of hydrophobicmaterial (e.g., a hydrophobic coating), such as apolytetrafluoroethylene (PTFE) layer (e.g., a PTFE tape). For example,the polytetrafluoroethylene (PTFE) matrix is patterned on a glass slide(e.g., a microscope slide) so that the PTFE matrix covers portions ofthe glass microscope slide and the remaining portions of the glassmicroscope slide are not covered by the PTFE matrix. The PTFE matrix hashydrophobic characteristics and the portions of the glass microscopeslide that are not covered by the PTFE matrix have hydrophiliccharacteristics. Aqueous solutions that include samples (e.g., cells)are typically placed on hydrophilic areas of the slide.

FIG. 5B illustrates a partial cross-section of array plate 522. In arrayplate 522, secondary areas 514 and 516 are offset from the plane onwhich surrounding hydrophobic area 506 is located, and primary area 512is offset from the plane on which secondary areas 514 and 516 arelocated (e.g., primary area 512 is located at a primary area depth fromsurrounding hydrophobic area 506, secondary areas 514 and 516 arelocated at a secondary area depth from surrounding hydrophobic area 506,and the first region depth is greater than the secondary area depth).

FIG. 5C illustrates a partial cross-section of array plate 524. In arrayplate 524, secondary areas 514 and 516 are located on a same plane assurrounding hydrophobic area 506. Primary area 512 is located offsetfrom the plane on which secondary areas 514 and 516 are located (e.g.,primary area 512 protrudes from secondary areas 514 and 516).

FIG. 5D illustrates a partial cross-section of array plate 526. In arrayplate 526, secondary areas 514 and 516 are offset from the plane onwhich surrounding hydrophobic area 506 is located. Primary area 512 isoffset from the plane on which secondary areas 514 and 516 are located(e.g., primary area 512 is located at a primary area depth fromsurrounding hydrophobic area 506, secondary areas 514 and 516 arelocated at a secondary area depth from surrounding hydrophobic area 506,and the first region depth is less than the secondary area depth). Insome embodiments, primary area 512 is located on a plane on whichsurrounding hydrophobic area 506 is located (e.g., the first regiondepth is zero).

Although FIGS. 5A-5D illustrate dispenser 210 and aspirator 220 to showthe positioning of dispenser 210 over secondary area 514 and aspirator220 over secondary area 516, dispenser 210 and aspirator 220 are notpart of the array plate.

FIG. 5E illustrates a partial cross-section of array plate 528. Arrayplate 528 is similar to array plate 526 shown in FIG. 5D, except thatarray plate 528 includes walls 518.

In some embodiments, as shown in FIG. 5D, walls 518 are separated fromhydrophilic regions (e.g., primary area 512 and secondary areas 514 and516) by hydrophobic region 506. In some embodiments, walls 518 extendsdirectly from the hydrophilic regions.

In some implementations, walls 518 define a well so that the well canhold a larger volume of liquid than a volume of liquid that the primaryarea and the secondary areas can hold. This allows washing with a largervolume of wash liquid, thereby enhancing the efficiency of washing.

In some embodiments, walls 518 are made of hydrophobic material (e.g.,polytetrafluoroethylene). In some embodiments, walls 518 are made ofhydrophilic material.

In some embodiments, walls 518 are positioned away from the primaryregion 512 by a predefined distance (e.g., at least 1 mm, at least 2 mm,at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7mm, at least 8 mm, at least 9 mm, at least 10 mm, etc.). This reducesthe likelihood that cells get into the corner around walls 518.

In some embodiments, walls 518 are configured to removably couple (e.g.,mate) with cap 530, as shown in FIG. 5E. Cap 530 is placed over walls518 to preventing spillage of a liquid located on array plate 528 (e.g.,a sample solution or mixture 508). For example, cap 530 is placed overwalls 518 for transportation of array plate 528 and/or shaking oragitation of array plate 528. In some embodiments, walls 518 and/or cap530 have mechanical features for maintaining cap 530 in place (e.g.,walls 518 and cap 530 have mating threads, or cap 530 has latches andwalls 518 have corresponding indentations for preventing slippage oflatches).

Although array plate 528 shown in FIG. 5E is based on array plate 526,any other array plate (including the array plates illustrated herein,such as array plate 520, 522, 524, and 526) can be modified to havewalls 518. In some embodiments, walls 518 are integrated with base 502.In some embodiments, walls 518 are formed separate from base 502 andsubsequently attached to base 502 (e.g., removable grids 552 shown inFIG. 5N).

FIG. 5F illustrates a partial cross-section of array plate 532. Arrayplate 532 is similar to array plate 522 shown in FIG. 5B, except thatregion 542 around primary area 512 is filleted (e.g., edge(s) aroundprimary area 512 has a rounded corner). In some embodiments, the regionaround primary area 512 (e.g., region 542) is chamfered (e.g., edge(s)around primary area 512 has a beveled corner). In some embodiments, thechamfered region is formed by removing material from array plate 532. Insome embodiments, the chamfered region is formed by molding (e.g.,injection molding).

Using array plate 532 with filleted or chamfered corners reduces cellsremaining adjacent to the corners (after washing), thereby reducestrapping of one or more liquid droplets by the cells located adjacent tothe corners (e.g., by capillary force between the cells and the platesurface around the corners). Thus, array plate 532 improves theefficiency of washing. In some embodiments, a radius of curvature (of arounded corner) is at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, or 1mm. For example, the radius of curvature is 0.5 mm, 0.6 mm, 0.7 mm, 0.8mm, or 0.9 mm. In some embodiments, a chamfer width is at least 0.1 mm,0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, or 1 mm. For example, the chamfer widthis 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, or 0.9 mm. In some embodiments, achamfer depth is at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, or 1mm. For example, the chamfer depth is 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, or0.9 mm. In some embodiments, a chamfer length is at least 0.14 mm, 0.28mm, 0.42 mm, 0.56 mm, 0.7 mm, or 1.4 mm. For example, the chamfer lengthis 0.7 mm, 0.84 mm, 0.98 mm, 1.12 mm, or 1.26 mm.

FIG. 5G illustrates a partial cross-section of array plate 534. Arrayplate 534 is similar to array plate 532 shown in FIG. 5F, except thatregions 544 around secondary areas 514 and 516 are filleted (e.g., edgesaround second regions 514 and 516 have a rounded corner). In someembodiments, the regions around secondary areas 514 and 516 (e.g.,regions 544) are chamfered (e.g., edges around secondary areas 514 and516 have beveled corners). In some embodiments, the chamfered regionsare formed by removing material from array plate 534. In someembodiments, the chamfered regions are formed by molding (e.g.,injection molding).

FIG. 5H illustrates a partial cross-section of array plate 536. Arrayplate 536 is similar to array plate 534 shown in FIG. 5G, except that aplurality of structures is defined in primary area 512, the plurality ofstructures configured to retain cells in the sample solution or mixture508 during dispensing of liquid from dispenser 210 and/or aspiration ofthe sample solution or mixture with aspirator 220. In some embodiments,the plurality of structures includes an array of dimples. In someembodiments, a respective dimple has a characteristic dimension (e.g., adiameter, width, depth, etc.) between 1 and 100 μm (e.g., ahalf-spherical dimple having a diameter of 20 μm). In some embodiments,a respective dimple has a characteristic dimension between 10 and 50 μm.In some embodiments, the dimples have an asymmetric shape (e.g., a righttriangular cross section, as shown in FIG. 5H) so that the dimples canretain the cells better when the liquid flows from dispenser 210 toaspirator 220.

FIG. 5I illustrates a partial cross-section of array plate 538. Arrayplate 538 is similar to array plate 534 shown in FIG. 5G, except that aplurality of structures 568 is located over at least primary area 512.In some embodiments, the plurality of structures 568 has a shape ofpillars. In some embodiments, a plurality of structures 568 has a shapeof claws as shown in FIG. 5I. In some embodiments, the plurality ofstructures 568 includes a magnetic material (e.g., a ferromagneticmaterial), and the plurality of structures 568 is held by a magneticforce (e.g., a magnetic force induced by a magnetic field from magnet572 located below array plate 538). In some embodiments, array plate 538includes magnet 572. In some embodiments, magnet 572 is removablycoupled with array plate 538.

FIGS. 5J-5K illustrate an example array plate in accordance with someembodiments.

FIG. 5J is a perspective view of a portion of the array plate inaccordance with some embodiments. The portion of the array plate shownin FIG. 5G has primary area 512 surrounded by region 542. As explainedabove with respect to FIG. 5F, region 542 is filleted or chamfered. Insome embodiments, region 542 is filleted and has a radius of curvature(R1) that corresponds to at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm. In someembodiments, region 542 has a radius of curvature (R1) that is less than0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm,1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7mm, 8 mm, 9 mm, or 10 mm. In some embodiments, the region 542 has aradius of curvature that is between 0.1 mm and 1 mm. In someembodiments, the region 542 has a radius of curvature that is between0.2 mm and 0.8 mm.

The portion of the array plate shown in FIG. 5J also has secondary areas514 and 516. In FIG. 5J, a respective second region (e.g., secondaryarea 514 or secondary area 516) is surrounded by region 544. In someembodiments, region 544 is filleted or chamfered. In some embodiments,region 544 is filleted and has a radius of curvature (R3) thatcorresponds to at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm,0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 3mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm. In some embodiments,region 544 has a radius of curvature (R3) that is less than 0.1 mm, 0.2mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.2mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9mm, or 10 mm. In some embodiments, the region 544 has a radius ofcurvature that is between 0.1 mm and 1 mm. In some embodiments, theregion 544 has a radius of curvature that is between 0.2 mm and 0.8 mm.In some embodiments, the radius of curvature (R3) of region 544 is lessthan the radius of curvature (R1) of region 542.

FIG. 5J also illustrates that an edge of secondary area 514 facingprimary area 512 has a radius of curvature (R2) in some embodiments. Insome embodiments, the radius of curvature (R2) corresponds to at least0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm,1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7mm, 8 mm, 9 mm, or 10 mm. In some embodiments, the radius of curvature(R2) is less than 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7mm, 0.8 mm, 0.9 mm, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 3 mm, 4mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm. In some embodiments, theradius of curvature (R2) is between 0.1 mm and 1 mm. In someembodiments, the radius of curvature (R2) is between 0.2 mm and 0.8 mm.In some embodiments, the radius of curvature (R2) is identical to theradius of curvature (R1) of region 512. In some embodiments, the radiusof curvature (R2) is identical to the radius of curvature (R3) of region544.

FIG. 5K is a partial cross-sectional view of the array plate shown inFIG. 5J.

The array plate shown in FIGS. 5J-5K have rounded corners for bothprimary and secondary areas, which further improves the efficiency ofwashing operations.

FIG. 5L illustrates an array plate in accordance with some embodiments.

The array plate shown in FIG. 5L is similar to the array plate shown inFIG. 5K, except that the array plate shown in FIG. 5L defines two ormore channels 562 and 564. In some embodiments, channel 562 isconfigured to introduce or provide liquid (e.g., a wash liquid, areagent liquid, etc.) to the array plate. In some embodiments, channel562 is configured to couple with a dispenser (e.g., with a coupler, suchas a clip, configured to removably couple with a nozzle of thedispenser). In some embodiments, channel 564 is configured to removeliquid (e.g., a sample solution or a mixture of the sample solution withother liquids) on the array plate. In some embodiments, channel 564 isconfigured to couple with an aspirator (e.g., with a coupler, such as aclip, configured to removably couple with a nozzle of the aspirator). Insome embodiments, secondary areas 514 and 516 are offset from eachother. For example, secondary area 514 is located on a first plane andsecondary area 516 is located on a second plane that is located awayfrom the first plane (e.g., the second plane is located below the firstplane).

FIG. 5M illustrates an array plate in accordance with some embodiments.

The array plate shown in FIG. 5M is similar to the array plate shown inFIG. 5L, except that the array plate shown in FIG. 5M defines only onechannel 564.

In some embodiments, channel 564 is configured to remove liquid (e.g., asample solution or a mixture of the sample solution with other liquids)on the array plate. In some embodiments, similar to the array plateshown in FIG. 5M, channel 564 is configured to couple with an aspirator(e.g., with a coupler, such as a clip, configured to removably couplewith a nozzle of the aspirator). In such embodiments, liquid (e.g., awash liquid or a reagent solution) is introduced by using dispenser 210that is located above the array plate. In some embodiments, secondaryarea 516 is located on a first plane and primary area 512 is located ona second plane that is located away from the first plane (e.g., thesecond plane is located below the first plane). In some embodiments thefirst plane is separated from the second plane by a predefined distance(e.g., a well height that is selected for improved cell retention andwashing efficiency).

In some embodiments, channel 564 is configured to introduce or provideliquid (e.g., a wash liquid, a reagent liquid, etc.) to the array plate.In some embodiments, channel 564 is configured to couple with adispenser (e.g., with a coupler, such as a clip, configured to removablycouple with a nozzle of the dispenser). In such embodiments, liquid(e.g., a sample solution or a mixture of the sample solution withanother liquid) is removed by using an aspirator that is located abovethe array plate.

FIG. 5N illustrates grid 552 configured for use with an example arrayplate in accordance with some embodiments. In some embodiments, grid 552includes polytetrafluoroethylene (PTFE). In some embodiments, grid 552is made of polytetrafluoroethylene (PTFE). Grid 552 defines one or morewalls. Gris 552 is configured for placement over the array plate todefine one or more wells (e.g., a respective well for each hydrophilicregion). Grid 552 increases a volume of liquid that can be placed on, oraround, each hydrophilic area. In some embodiments, elastomeric layer554 is positioned between grid 552 and the array plate. This, in somecases, reduces wicking of the liquid into the gap between grid 552 andthe array plate. In some embodiments, the hydrophobic area of the arrayplate is coated with a hydrophobic liquid immiscible so that wicking ofa hydrophilic liquid (e.g., water) is reduced or prevented.

FIG. 6A shows results of washing operations performed with aconventional micro-titer plate in accordance with some embodiments. Eachsample was washed with a four-fold dilution in each step, by leaving 25μl of the mixture and adding 75 μl of a fresh wash buffer.

The first three rows of the micro-titer plate show results of a beadretention test. A solution containing micro beads was washed six times.As shown in the image, the beads in the micro-titer plate tend tocluster along the edge of the well, which can reduce the assayperformance.

The next three rows (e.g., the fourth, fifth, and sixth rows) of themicro-titer plate show results of a wash efficiency test. A solutioncontaining a predefined concentration of an ink was washed multipletimes. The third column represents solutions after the first wash, thefourth column represents solutions after the second wash, the fifthcolumn represents solutions after the third wash, the sixth columnrepresents solutions after the fourth wash, the seventh columnrepresents solutions after the fifth wash, and the eighth columnrepresents solutions after the sixth wash. With the conventionalmicro-titer plate, the color of the ink is still visible even after sixwashes. This may be due to a remaining mixture clinging onto the edge bycapillary force. In addition, the variation in the color indicates thevariation in the wash efficiency from well to well. Such variation inthe color will lead to variations and errors in assay results.

FIG. 6B shows results of washing operations performed with the methoddescribed herein. Again, each sample was washed with a four-folddilution in each step, by leaving 25 μl of the mixture and adding 75 μlof a fresh wash buffer.

Each row represents sample washing performed at different nozzle depths.The first column represents solutions after the first wash, the secondcolumn represents solutions after the second wash, the third columnrepresents solutions after the third wash, the fourth column representssolutions after the fourth wash, the fifth column represents solutionsafter the fifth wash, and the sixth column represents solutions afterthe sixth wash. As shown in FIG. 6B, each solution has turned clearafter the sixth wash. Compared to the wash results shown in FIG. 6A,FIG. 6B shows that the washing efficiency of the method described hereinis far superior. In addition, the variation from sample to sample isreduced compared to the variation observed in FIG. 6A.

In addition, FIGS. 6C and 6D show that the method described hereinfacilitates cell sorting. FIG. 6C illustrates the results offluorescence-activated cell sorting of lymphocytes washed using aconventional method. FIG. 6D illustrates the results offluorescence-activated cell sorting of lymphocytes washed using themethod described herein. As shown in FIGS. 6C and 6D, the resolution offluorescence-activated cell sorting and the data quality have beenimproved by using the washing method described herein. Furthermore, byutilizing the array plates described herein, less time was required forpreparing a sample for flow cytometry analysis (as compared to usingconventional methods).

FIGS. 7A and 7B are microscope images of beads on array plates inaccordance with some embodiments. FIG. 7A shows results of washingoperations performed with an array plate without filleted corners (e.g.,array plates with sharp corners) in accordance with some embodiments,and FIG. 7B shows results of washing operations performed with an arrayplate with filleted corners (e.g., FIGS. 5F-5K) in accordance with someembodiments. For each array plate, we dispensed a solution containing 7μm diameter polystyrene beads (in order to simulate retention of cells)over a secondary area, shook the array plate with the solution using ashaker, and left the array plate to allow the polystyrene beads tosettle. Subsequently, the solution was aspirated slowly to remove thesolution while maintaining most of the polystyrene beads on the arrayplate. Thereafter, we washed the array plate by adding a flow cytometry(FACS) buffer, shaking the array plate, and aspirating the FACS buffer.We repeated these washing steps (e.g., repeat once, repeat twice, repeatthrice, etc.). Finally, we dispensed a phosphate buffered saline (PBS)solution, shook the array plate, and observed the array plate (e.g.,around the primary areas) using a microscope.

As shown in FIG. 7A, washing operations performed with an array platewithout filleted corners leave a large number of beads adjacent to thecorners (e.g., a periphery of the primary area). As shown in FIG. 7B,washing operations performed with an array plate with filleted cornersreduce the number of beads remaining adjacent to the corners, whichimproves the washing efficiency. In addition, fewer washing steps arerequired with the array plate with filleted corners than with the arrayplate without filleted corners.

Similarly, FIGS. 7C and 7D show that washing operations performed withan array plate without filleted corners (e.g., array plates having sharpcorners) have a large number of cells adjacent to the corners (FIG. 7C)and washing operations performed with an array plate with filletedcorners reduce the number of cells remaining adjacent to the corners(FIG. 7D).

In light of these principles, we turn to certain embodiments.

In accordance with some embodiments, an apparatus for washing an arrayplate includes one or more dispensers (e.g., dispenser 230 in FIG. 3A).A respective dispenser of the one or more dispensers is configured todispense a first liquid on the array plate (e.g., dispenser 230 isconfigured to dispense wash liquid 106 on the array plate). Therespective dispenser includes a first piston configured to slide atleast partially within a first channel (e.g., piston 236 is configuredto slide within channel 234); and a first valve (e.g., valve 212)configured to allow the first liquid in the first channel to bedispensed from the first channel through the first valve and prevent aliquid from entering into the first channel through the first valve.

In some embodiments, the respective dispenser includes a first tubedefining the first channel and configured for holding the first liquid(e.g., tube 235 defining channel 234). The first piston is slidablycoupled with the first tube (e.g., piston 236 is configured to slidewithin tube 235 while piston 236 remains in contact with tube 235). Thefirst valve is coupled with the first tube and configured to allow thefirst liquid in the first tube to be dispensed from the first tubethrough the first valve and prevent a liquid from entering into thefirst tube through the first valve.

In some embodiments, the first piston defines a second channel (e.g.,channel 238) that is distinct from the first channel. The respectivedispenser also includes a second valve (e.g., valve 252) that isdistinct from the first valve. The second valve is configured to allowthe first liquid in the second channel to be dispensed from the secondchannel through the second valve and prevent a liquid from entering intothe second channel through the second valve.

In some embodiments, the first piston comprises a second tube (e.g., theshell of piston 236) that defines the second channel. In someembodiments, the second tube is distinct from the first tube. The secondtube is configured for holding the first liquid. The second valve iscoupled with the second tube and configured to allow the first liquid inthe second tube to be dispensed from the second tube through the secondvalve and prevent a liquid from entering into the second tube throughthe second valve.

In some embodiments, the apparatus further includes one or moreaspirators (e.g., aspirator 240). A respective aspirator of the one ormore aspirators is configured to aspirate a liquid on the array plate.The respective aspirator includes a second piston (e.g., piston 246)configured to slide at least partially within a third channel (e.g.,channel 244); and a third valve (e.g., valve 222) configured to allowthe liquid on the array plate to be aspirated into the third channelthrough the third valve and prevent a liquid in the third channel fromexiting from the third channel through the third valve.

In some embodiments, the respective aspirator includes a third tube(e.g., tube 245) defining the third channel. The third tube is distinctfrom the first tube. The second piston is slidably coupled with thethird channel. (e.g., piston 246 is configured to slide within channel244). The third valve is coupled with the third tube and configured toallow the liquid on the array plate to be aspirated into the thirdchannel through the third valve and prevent a liquid from entering intothe third tube through the third valve.

In some embodiments, the second piston defines a fourth channel (e.g.,channel 248) that is distinct from the third channel. The respectiveaspirator also includes a fourth valve (e.g., valve 262) that isdistinct from the third valve. The fourth valve is configured to allowthe liquid in the third channel to enter into the fourth channel andprevent a liquid in the fourth channel from exiting from the fourthchannel through the fourth valve.

In some embodiments, the second piston comprises a fourth tube (e.g.,the shell of piston 246) that defines the fourth channel. In someembodiments, the fourth tube is distinct from the third tube. The fourthvalve is coupled with the fourth tube and configured to allow the liquidin the third tube to enter into the fourth tube through the fourth valveand prevent a liquid in the fourth tube from exiting from the fourthtube through the fourth valve.

In some embodiments, the one or more dispensers comprise a plurality ofdispensers, which is arranged in a first array (e.g., dispensers 210 inFIG. 4F). In some embodiments, the one or more dispenses comprise aplurality of aspirators, which is arranged in a second array (e.g.,aspirators 220 in FIG. 4F).

In some embodiments, the plurality of dispensers is arranged in atwo-dimensional array having multiple rows and multiple columns ofdispensers (e.g., FIG. 4G). In some embodiments, the plurality ofaspirators is arranged in a two-dimensional array having multiple rowsand multiple columns of aspirators (e.g., FIG. 4G).

In some embodiments, two or more first channels of the plurality ofdispensers are defined in a first block (e.g., the cylinder block inFIG. 3H).

In some embodiments, two or more second channels of the plurality ofdispensers are defined in a second block (e.g., the dispenser pistonblock in FIG. 3H). In some embodiments, the first pistons are integratedwith the second block. In some embodiments, the second block is distinctfrom the first block.

In some embodiments, two or more third channels of the plurality ofaspirators are defined in a first block (e.g., the cylinder block inFIG. 3H).

In some embodiments, two or more fourth channels of the plurality ofaspirators are defined in a third block (e.g., the aspirator pistonblock in FIG. 3H). In some embodiments, the second pistons areintegrated with the third block. In some embodiments, the third block isdistinct from the first block. In some embodiments, the third block isdistinct from the second block.

In accordance with some embodiments, an apparatus for washing an arrayplate includes one or more dispensers, a respective dispenser of the oneor more dispensers configured to dispense a first liquid on the arrayplate; and one or more aspirators that are distinct from the one or moredispensers. A respective aspirator of the one or more aspiratorsincludes a positive displacement pump configured to aspirate liquid onthe array plate. In some embodiments, the positive displacement pump isconfigured to aspirate a predefined or preselected volume of the liquidon the array plate.

In accordance with some embodiments, an apparatus is configured forwashing an array plate having a primary area and at least two secondaryareas. The apparatus includes a first set of one or more pipettesconfigured to dispense a first liquid at a first time on a firstsecondary area of the array plate and a second set of one or morepipettes that is distinct from (and mutually exclusive to) the first setof one or more pipettes, the second set of one or more pipettesconfigured to aspirate liquid on the array plate from a second secondaryarea of the array plate at either the first time or a second time thatis distinct from the first time (e.g., the second time is subsequent tothe first time). The first set of one or more pipettes is alsoconfigured to aspirate at either the second time or a third time that isdistinct from the second time (e.g., the third time is subsequent to thesecond time) the liquid on the array plate from the first secondaryarea. For example, in some cases, the first set of one or more pipettes(e.g., one or more dispensers) are used to dispense the first liquid ata first time at a location adjacent to the first secondary area of thearray plate, and thereafter, the second set of one or more pipettes(e.g., one or more aspirators) are used to aspirate a first portion ofliquid on the array plate from the second secondary area of the arrayplate. Subsequently, the first set of one or more pipettes are used toaspirate a second portion of the liquid on the array plate from thefirst secondary area of the array plate. Dispensing the first liquid ata location adjacent to the first secondary area pushes away any cellslocated, before dispensing the first liquid, on the first secondary areaof the array plate from the first secondary area of the array plate(e.g., toward the primary area), and thus, reduces a number of cellsthat can be aspirated (and thus, gets lost) in a subsequent aspirationfrom a location adjacent to the first secondary area. This allows alarger portion of the liquid to be aspirated without loss (or withreduced loss) of cells through aspiration.

In accordance with some embodiments, a method for washing an array platehaving a primary area and at least two secondary areas includesdispensing, with a first set of one or more pipettes, a first liquid ata first time on a first secondary area of the array plate. The methodalso includes aspirating, with a second set of one or more pipettes thatis distinct from (and mutually exclusive to) the first set of one ormore pipettes, liquid on the array plate from a second secondary area ofthe array plate at either the first time or a second time that isdistinct from the first time (e.g., the second time is subsequent to thefirst time). The method further includes aspirating, with the first setof one or more pipettes (or a third set of one or more pipettes that isdistinct from, and mutually exclusive to, the first set of one or morepipettes and the second set of one or more pipettes) at either thesecond time or a third time that is distinct from the second time (e.g.,the third time is subsequent to the second time) the liquid on the arrayplate from the first secondary area.

In accordance with some embodiments, a method for washing a sampleincludes obtaining an array plate that includes an array of hydrophilicareas surrounded by one or more hydrophobic areas. A respective solutioncontaining a sample is located on a respective hydrophilic area of thearray of hydrophilic areas. The respective hydrophilic area includes oneor more indentations from a respective surrounding hydrophobic area ofthe one or more hydrophobic areas. The respective hydrophilic areaincludes a first indented surface (e.g., secondary area 514 and/orsecondary area 516) that is offset from a reference surface defined bythe respective surrounding hydrophobic area. The method also includesplacing an aspirator nozzle above the respective hydrophilic area atleast 100 μm from the first indented surface; and aspirating thesolution with the aspirator nozzle while the aspirator nozzle is locatedat least 100 μm from the first indented surface (e.g., FIG. 2A).

In some embodiments, the solution is aspirated with the aspirator nozzlewhile the aspirator nozzle is located at least 200 μm from the firstindented surface. In some embodiments, the solution is aspirated withthe aspirator nozzle while the aspirator nozzle is located at least 300μm from the first indented surface.

In some embodiments, the first indented surface is offset from thereference surface by a first distance; and the respective hydrophilicarea includes a second indented surface that is offset from thereference surface by a second distance.

In some embodiments, the second distance is distinct from the firstdistance (e.g., FIG. 5B). In some embodiments, the second distance isgreater than the first distance. In some embodiments, the first distanceis greater than the second distance.

In some embodiments, the second distance is equal to the first distance.

In some embodiments, the second distance is less than 3000 μm. In someembodiments, the second distance is 2000 μm or less. In someembodiments, the second distance is 1750 μm or less. In someembodiments, the second distance is 1500 μm or less. In someembodiments, the second distance is 1250 μm or less. In someembodiments, the second distance is 1000 μm or less. In someembodiments, the second distance is 750 μm or less. In some embodiments,the second distance is 500 μm or less.

In some embodiments, the first distance is 1000 μm or less. In someembodiments, the first distance is 750 μm or less. In some embodiments,the first distance is 500 μm or less. In some embodiments, the firstdistance is 250 μm or less.

In some embodiments, the method includes placing the aspirator nozzleabove the respective hydrophilic area at least 100 μm from the firstindented surface. In some embodiments, the method includes placing theaspirator nozzle above the respective hydrophilic area at least 200 μmfrom the first indented surface. In some embodiments, the methodincludes placing the aspirator nozzle above the respective hydrophilicarea at least 300 μm from the first indented surface.

In some embodiments, the solution is aspirated at a rate between 1 and50 μl/sec. In some embodiments, the solution is aspirated at a ratebetween 2 and 20 μl/sec. In some embodiments, the solution is aspiratedat a rate of 20 μl/sec or less. In some embodiments, the solution isaspirated at a rate of 10 μl/sec or less. In some embodiments, thesolution is aspirated at a rate of 5 μl/sec or less.

In some embodiments, the method includes, prior to aspirating therespective solution with the aspirator nozzle, shaking the array plate.Shaking the array plate facilitates mixing of the wash liquid and thesample solution. In some cases, shaking the array plate also facilitatesreleasing chemical and/or biological reagents from the surface, therebyimproving removal of such chemical and/or biological reagents.

In some embodiments, the method includes, subsequent to shaking thearray plate and prior to aspirating the respective solution with theaspirator nozzle, settling the sample in the respective solution by morethan 10 minutes. In some embodiments, the method includes, subsequent toshaking the array plate and prior to aspirating the respective solutionwith the aspirator nozzle, settling the sample in the respectivesolution by more than 15 minutes.

In some embodiments, the method includes, subsequent to shaking thearray plate and prior to aspirating the respective solution with theaspirator nozzle, settling the sample in the respective solution by lessthan 90 minutes. In some embodiments, the method includes, subsequent toshaking the array plate and prior to aspirating the respective solutionwith the aspirator nozzle, settling the sample in the respectivesolution by less than 60 minutes.

In some embodiments, the respective solution has a volume less than 200Ht. In some embodiments, the respective solution has a volume less than70 Ht.

In some embodiments, the method includes introducing a liquid (e.g., awash liquid, a reagent liquid, etc.) to the solution (e.g., dispensingthe liquid onto the solution). In some embodiments, the liquid isintroduced at a rate between 1 and 50 μl/sec. In some embodiments, theliquid is introduced at a rate between 2 and 20 μl/sec. In someembodiments, the liquid is introduced at a rate of 20 μl/sec or less. Insome embodiments, the liquid is introduced at a rate of 10 μl/sec orless. In some embodiments, the liquid is introduced at a rate of 5μl/sec or less.

In some embodiments, the operation of introducing the liquid and theoperation of aspirating the solution are repeated at least three times.In some embodiments, the operation of introducing the liquid and theoperation of aspirating the solution are repeated no more than ninetimes.

In accordance with some embodiments, a method for washing a sampleincludes obtaining an array plate that includes an array of hydrophilicareas surrounded by one or more hydrophobic areas. A respective solutioncontaining a sample is located on a respective hydrophilic area of thearray of hydrophilic areas. The respective hydrophilic area includes oneor more indentations from a respective surrounding hydrophobic area ofthe one or more hydrophobic areas. The respective hydrophilic areaincludes at least two indented surfaces (e.g., secondary area 514 andsecondary area 516) that are offset from a reference surface defined bythe respective surrounding hydrophobic area. The method also includesdispensing a first liquid (e.g., a wash liquid) onto a first indentedsurface of the array plate with a first set of one or more pipettes at afirst time and aspirating liquid on the array plate from a secondindented surface of the array plate with a second set of one or morepipettes distinct from the first set of one or more pipettes at thefirst time or a second time that is distinct from the first time. Insome embodiments, the method further includes aspirating the liquid onthe array plate from the first indented surface of the array plate withthe first set of one or more pipettes at the second time.

In accordance with some embodiments, an apparatus is configured forperforming any method described herein.

In some embodiments, the apparatus includes one or more dispensers, arespective dispenser of the one or more dispensers configured todispense a first liquid on an array plate. The respective dispenserincludes a first piston configured to slide at least partially within afirst channel; and a first valve configured to allow the first liquid inthe first channel to be dispensed from the first channel through thefirst valve and prevent a liquid from entering into the first channelthrough the first valve.

In accordance with some embodiments, a device for washing a sampleincludes a plate having an array of hydrophilic areas; and one or morehydrophobic areas surrounding the array of hydrophilic areas. Arespective hydrophilic area of the array of hydrophilic areas is offsetfrom a surrounding hydrophobic area of the one or more hydrophobicareas. The respective hydrophilic area includes a primary area and twoor more secondary areas that extend from the primary area on a planedefined by the primary area.

In some embodiments, the surrounding hydrophobic area is coated withhydrophobic oil.

In some embodiments, the primary area has a shape of a circle and eachsecondary area of the two or more secondary areas has a shape of apartial circle.

In some embodiments, the primary area is located on a first plane, afirst secondary area of the two or more secondary areas is located on asecond plane that is offset from the first plane, and a second secondaryarea of the two or more secondary areas is located on a third plane thatis offset from the first plane. In some embodiments, the second planeand the third plane overlap each other. In some embodiments, the secondplane is offset from the third plane.

In some embodiments, the device defines a first channel (e.g., channel562, FIG. 5L) with a through-hole extending from a first secondary areaof the two or more secondary areas to a bottom of the device so thatliquid can be transported through the first channel between the bottomof the device and the first secondary area.

In some embodiments, the device defines a second channel (e.g., channel564, FIG. 5L) that is distinct from the first channel and includes athrough-hole extending from a first secondary area of the two or moresecondary areas to a bottom of the device so that liquid can betransported through the first channel between the bottom of the deviceand the second secondary area. In some embodiments, the device definesthe first channel without defining the second channel (e.g., FIG. 5M).

In some embodiments, a plurality of structures is defined on the primaryarea (e.g., FIG. 5H).

In some embodiments, a plurality of structures is located on the primaryarea (e.g., Figure SI).

In some embodiments, the plurality of structures includes a magneticmaterial so that the plurality of structures can be held with a magneticforce.

Various aspects and characteristics of the methods of using the arrayplates described above are applicable to array slides (e.g., adding oneor more solutions to one or more liquid droplets of the respectiveliquid droplets, performing an immunoassay, and washing a respectiveliquid droplets), and vice versa. Because these aspects andcharacteristics are described above, they are not repeated herein forbrevity.

It is well known to a person having ordinary skill in the art that arrayslides and plates can be used in many other biological and chemicalreactions. Therefore, such details and specific examples are omitted forbrevity.

The foregoing description, for purpose of explanation, has beendescribed with reference to specific embodiments. However, theillustrative discussions above are not intended to be exhaustive or tolimit the embodiments to the precise forms disclosed. Many modificationsand variations are possible in view of the above teachings. Theembodiments were chosen and described in order to best explain theprinciples of the invention and its practical applications, to therebyenable others skilled in the art to best utilize the invention andvarious embodiments with various modifications as are suited to theparticular use contemplated.

What is claimed is:
 1. A method for washing a sample, the methodcomprising: obtaining an array plate that includes an array ofhydrophilic areas surrounded by one or more hydrophobic areas, wherein:a respective solution containing a sample is located on a respectivehydrophilic area of the array of hydrophilic areas; the respectivehydrophilic area includes one or more indentations from a respectivesurrounding hydrophobic area of the one or more hydrophobic areas; andthe respective hydrophilic area includes a first indented surface thatis offset from a reference surface defined by the respective surroundinghydrophobic area; placing an aspirator nozzle above the respectivehydrophilic area at least 100 μm from the first indented surface; andaspirating the solution with the aspirator nozzle while the aspiratornozzle is located at least 100 μm from the first indented surface. 2.The method of claim 1, wherein: the first indented surface is offsetfrom the reference surface by a first distance; and the respectivehydrophilic area includes a second indented surface that is offset fromthe reference surface by a second distance that is distinct from thefirst distance.
 3. The method of claim 2, wherein: the second distanceis less than 3000 μm.
 4. The method of claim 1, including: placing theaspirator nozzle above the respective hydrophilic area at least 300 μmfrom the first indented surface.
 5. The method of claim 1, wherein: thesolution is aspirated at a rate of 20 μl/sec or less.
 6. The method ofclaim 5, wherein: the solution is aspirated at a rate of 5 μl/sec orless.
 7. The method of claim 1, including: prior to aspirating therespective solution with the aspirator nozzle, shaking the array plate.8. The method of claim 7, including: subsequent to shaking the arrayplate and prior to aspirating the respective solution with the aspiratornozzle, settling the sample in the respective solution by more than 10minutes.
 9. The method of claim 7, including: subsequent to shaking thearray plate and prior to aspirating the respective solution with theaspirator nozzle, settling the sample in the respective solution by lessthan 90 minutes.
 10. The method of claim 1, wherein: the respectivesolution has a volume less than 200 Ht.
 11. The method of claim 1,including aspirating the solution with an aspirator that includes: anaspirator piston configured to slide at least partially within anaspirator channel; and an aspirator valve configured to allow a liquidon the array plate to be aspirated into the aspirator channel throughthe aspirator valve and prevent a liquid in the aspirator channel fromexisting from the aspirator channel through the aspirator valve.
 12. Themethod of claim 11, wherein: the aspirator piston defines an inneraspirator channel that is distinct from the aspirator channel; and theaspirator also includes an inner aspirator valve configured to allow theliquid in the aspirator channel to enter into the inner aspiratorchannel and prevent a liquid in the inner aspirator channel from exitingfrom the inner aspirator channel through the inner aspirator valve. 13.The method of claim 11, further comprising dispensing a liquid using adispenser is distinct from the aspirator and includes: a dispenserpiston configured to slide at least partially within a dispenserchannel; and a dispenser valve configured to allow a liquid in thedispenser channel to be dispensed from the dispenser channel through thedispenser valve and prevent a liquid from entering into the dispenserchannel through the dispenser valve.
 14. The method of claim 13,wherein: the dispenser piston defines an inner dispenser channel that isdistinct from the dispenser channel; and the dispenser also includes aninner dispenser valve configured to allow the liquid in the innerdispenser channel to enter into the dispenser channel and prevent aliquid in the dispenser channel from entering into the inner dispenserchannel through the inner dispenser valve.
 15. The method of claim 1,further comprising dispensing a liquid using a dispenser is distinctfrom the aspirator and includes: a dispenser piston configured to slideat least partially within a dispenser channel; and a dispenser valveconfigured to allow a liquid in the dispenser channel to be dispensedfrom the dispenser channel through the dispenser valve and prevent aliquid from entering into the dispenser channel through the dispenservalve.
 16. The method of claim 15, wherein: the dispenser piston definesan inner dispenser channel that is distinct from the dispenser channel;and the dispenser also includes an inner dispenser valve configured toallow the liquid in the inner dispenser channel to enter into thedispenser channel and prevent a liquid in the dispenser channel fromentering into the inner dispenser channel through the inner dispenservalve.
 17. An apparatus configured for performing the method of claim 1.18. The apparatus of claim 17, further comprising: one or moredispensers, a respective dispenser of the one or more dispensersconfigured to dispense a first liquid on an array plate, the respectivedispenser including: a first piston configured to slide at leastpartially within a first channel; and a first valve configured to allowthe first liquid in the first channel to be dispensed from the firstchannel through the first valve and prevent a liquid from entering intothe first channel through the first valve.
 19. The apparatus of claim18, wherein: the first piston defines a second channel that is distinctfrom the first channel; and the respective dispenser also includes asecond valve that is distinct from the first valve, wherein the secondvalve is configured to allow the first liquid in the second channel tobe dispensed from the second channel through the second valve andprevent a liquid from entering into the second channel through thesecond valve.
 20. A device for washing a sample, the device comprising:a plate having: an array of hydrophilic areas; and one or morehydrophobic areas surrounding the array of hydrophilic areas, wherein: arespective hydrophilic area of the array of hydrophilic areas is offsetfrom a surrounding hydrophobic area of the one or more hydrophobicareas; and the respective hydrophilic area includes a primary area andtwo or more secondary areas that extend from the primary area on a planedefined by the primary area.